Tagetes erecta marigolds with altered carotenoid compositions and ratios

ABSTRACT

A marigold plant, a regenerable portion thereof and seed are disclosed whose flower petals, leaves or flower petals and leaves contain one or more of an enhanced zeaxanthin ratio, an enhanced neoxanthin plus violaxanthin ratio, an enhanced β-carotene ratio, an enhanced α-cryptoxanthin ratio, an enhanced phytoene ratio or an enhanced phytofluene ratio relative to that ratio in a non-mutant marigold. The flower petals of such a plant also typically contain zeta-carotene that is not normally found in such petals. Also disclosed are methods of preparing such plants, oleoresins and comestible materials that have such carotenoid ratios.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Serial No. 60/302,460,filed Jun. 29, 2001.

TECHNICAL FIELD

[0002] The present invention relates to a marigold plant that containscarotenoid pigments present at other than the usual ratios. Theinvention more particularly relates to a marigold plant, a regenerableportion thereof, a hybrid or later generation whose petals, leaves orboth petals and leaves, contain an enhanced ratio of one or morecarotenoid compounds relative to lutein, and also seed that producessuch a marigold plant, an oleoresin produced from such flowers or leavesand comestible products made using zeaxanthin and lutein. The flowerpetals of such a contemplated marigold typically also contain ameasurable amount of zeta-carotene, a compound not normally found inmarigold flower petals.

BACKGROUND OF THE INVENTION

[0003] Numerous epidemiological studies in various populations haveshown that consumption of substantial amounts of fruits and vegetablesrich in carotenoids can reduce the risk of acquiring several types ofcancers. As a result, scientists have been focusing on investigating theprotective effect of carotenoids such as beta-(β-)carotene in preventionof cancer, cardiovascular and eye diseases. These studies have beencarried out despite the fact that β-carotene is only one of theprominent carotenoids found in fruits and vegetables whose consumptionhas been associated with health benefits. The reasons for such focus canbe attributed to the pro-vitamin A activity of β-carotene and thelimited commercial availability of other prominent food carotenoids.

[0004] Among the 40 to 50 carotenoids that are available from the dietand may be absorbed, metabolized, or utilized by the human body, only 13carotenoids and 12 of their stereoisomers are routinely found in humanserum and milk. [See Khachik et al., Anal. Chem., 69:1873-1881 (1997).]In addition, there are 8 carotenoid metabolites and one stereoisomer inhuman serum or plasma that result from a series of oxidation-reductionreactions of three dietary carotenoids: lutein, zeaxanthin and lycopene.These metabolites were first isolated and characterized by Khachik etal. [See Khachik et al., Anal. Chem., 64:2111-2122 (1992).]

[0005] In another study, the ingestion of purified supplements ofdietary (3R, 3′R, 6′R)-lutein and (3R, 3′R)-zeaxanthin was shown to notonly result in an increase in the blood levels of these compounds inhumans, but also in an increase in the concentration of their oxidativemetabolites in plasma. [See Khachik et al., J. Cellular Biochem., 22:236-246 (1995).] These findings provided preliminary evidence thatcarotenoids may function as antioxidants in disease prevention. Inaddition, these results also established the importance of non-vitaminA-active dietary carotenoids, particularly, lutein, zeaxanthin, andlycopene.

[0006] There is increasing evidence that the macular pigmentcarotenoids, lutein and zeaxanthin, may play an important role in theprevention of age-related macular degeneration (ARMD), cataractformation, and other light-induced oxidative eye damage. In 1985 and1993, Bone et al. demonstrated that the human macular pigment is acombination of lutein and zeaxanthin, and speculated that these dietarycarotenoids may play a role in the prevention of an eye disease ARMD.[See Bone et al., Vision Research, 25:1531-1535 (1985) and Bone et al.,Invest. Ophthalmol. Vis. Sci., 34: 2033-2040 (1993).] Further work in acase-controlled epidemiological study in which the high consumption offruits and vegetables, rich specifically in lutein and zeaxanthin wascorrelated to a 43 percent lower risk of ARMD later confirmed thatspeculation. [See Seddon et al., J. A. Med. Assoc., 272(18) 1413-1420(1994).] It has also been reported that an increased level of serumcarotenoids other than β-carotene is associated with a lower incidenceof heart disease. [See Morris et al., J. Amer. Med. Assoc.,272(18):1439-1441(1994).]

[0007] Bernstein et al. identified and quantified the dietarycarotenoids and their oxidative metabolites in all tissues of the humaneye and reported that nearly all ocular structures examined with theexception of vitreous, cornea and sclera had quantifiable levels ofdietary (3R, 3′R, 6′R)-lutein, zeaxanthin, their geometrical (E/Z)isomers, as well as their metabolites, (3R, 3′S, 6′R)-lutein(3′-epilutein) and 3-hydroxy-beta,epsilon-caroten-3′-one. In the iris,these pigments were thought likely to play a role in filteringphototoxic short-wavelength visible light and to act as antioxidant inthe ciliary body. Both mechanisms may be operative in the retinalpigment epithelium/choroid (RPE/choroids). [See Bernstein et al., Exp.Eye Research, 72(3): 215-223 (2001].]

[0008] A study of the distribution of macular pigment stereoisomers inthe human retina identified (3S, 3′S)-zeaxanthin in the adult retina,particularly in the macula. It was proposed that dietary lutein andzeaxanthin are transported into an individual's retina in the sameproportions found in the blood serum, although the two pigments arepresent in the eye in ratios different from those found in the blood.Thus, zeaxanthin predominates over lutein by a ratio greater than 2:1 inthe foveal region, with the macular pigment optical density dropping bya factor of 100 and the zeaxanthin to lutein ratio reversing to about1:2. [See Bone et al., Invest. Ophthalmol. Vis. Sci., 29:843-849(1988).]Some lutein is converted into the non-dietary mesozeaxanthin primarilyin the macula. [See Bone et al., Exp. Eye Res., 64(2): 211-218 (1997).]Such reports lend support to the critical role of ocular carotenoids,and therefore to the importance of commercial production of dietarycarotenoids in general, and particularly lutein and zeaxanthin.

[0009] The Tagetes genus is a member of the family Compositae,alternatively known as Asteraceae, and comprises some thirty species ofstrongly scented annual or perennial herbs. Tagetes are native fromArizona and New Mexico to Argentina. [See Hortus Third A ConciseDictionary of Plants Cultivated in the United States and Canada,MacMillan Publishing Company (1976).] Cultivated genera include Tageteserecta, commonly referred to as African marigold, Tagetes patula,commonly referred to as French marigold, Tagetes erecta x patula,commonly referred to as Triploid marigolds, and Tagetes tenuifolia alsoknown as Tagetes signata or signet marigold.

[0010] A marigold inflorescence is a solitary head comprised of a densecluster of several hundred sessile or subsessile small flowers alsoknown as florets. Marigolds have radiate flower heads with outer rayflorets that are ligulate or strap-shaped around the central tubularshaped disk florets. Some forms of marigold flower heads have most oftheir disk flowers transformed into ray flowers and contain few, if any,disk flowers. Such flower heads are referred to as double-flowered.

[0011] The ray flowers or florets are often referred to as petals by laypersons who may also refer to the flower heads as flowers. For ease ofunderstanding, marigold flower heads will be referred to herein asflowers or flower heads, whereas the flower head-component flowers orflorets will be referred to as petals.

[0012] Cultivated marigolds possess showy flowers and are useful forornamental purposes. In addition, the genus is recognized as a sourcefor natural colorants, essential oils, and thiophenes. Dried marigoldpetals and marigold petal concentrates obtained from so-calledxanthophyll marigolds are used as feed additives in the poultry industryto intensify the yellow color of egg yolks and broiler skin. [SeePiccalia et al., Ind. Crops and Prod., 8:45-51 (1998).] The carotenoidsdesired in poultry tissues are a function of their dietaryconcentration, because poultry do not have the ability to synthesizecarotenoids de novo. [See Balnave et al., Asian-Australiasian J. AnimalSci., 9(5): 515-517 (1996).]

[0013] Xanthophyll marigolds differ in several characteristics fromornamental marigolds. First and foremost, xanthophyll marigolds are usedas an extractable source for carotenoids and have plant habits thatdiffer from ornamental marigolds. Ornamental marigolds typically growonly about 45 to about 60 cm from the ground, whereas xanthophyllmarigolds grow to about 65 to about 70 cm from the ground. Xanthophyllmarigolds grow in a bushier habit than do ornamental marigolds, and canbe grown as row crops whereas ornamental marigolds typically cannot.Xanthophyll marigolds are typically dark orange in color, whereasornamentals can be white, yellow, or orange in color, or can have mixedcolors, including mahogany colors due to the presence of anthocyaninpigments.

[0014] The pigmenting ability of marigold petal meal resides largely inthe oxygenated carotenoid fraction known as the xanthophylls, primarilylutein esters. [See Piccalia et al., Ind. Crops and Prod., 8:45-51(1998).] The xanthophyll zeaxanthin, also found in marigold petals, hasbeen shown to be effective as a broiler pigmenter, producing a highlyacceptable yellow to yellow-orange color. [See Marusich et al., PoultrySci., 55:1486-1494 (1976).] Of the xanthophylls, the pigments lutein andzeaxanthin are the most abundant in commercially available hybrids.Structural formulas for lutein and zeaxanthin are shown below.

[0015] Each of lutein and zeaxanthin contains one hydroxyl group in eachof their terminal ring structures, so that each molecule contains twohydroxyl groups. Lutein is believed to be biologically produced by twoseparate hydroxylations of α-carotene, whereas zeaxanthin is believed tobe biologically produced by two separate hydroxylations of β-carotene.Both α-carotene and β-carotene are understood to be formed by the actionof appropriate cyclase enzymes on γ-carotene, which itself is formed bycyclization of lycopene. Lycopene, γ-carotene, α-carotene and β-caroteneare each hydrocarbon carotenoids.

[0016]FIG. 1 shows a schematic representation of the biologicalsynthesis pathway for the production of lutein and zeaxanthin and laterproducts from phytoene, the first C₄₀ carotenoid in the pathway. Luteinand zeaxanthin are present in marigold petals primarily as mono- anddi-esters of fatty acids. FIG. 1 also notes epoxide-containing laterproducts that can arise from zeaxanthin, of which violaxanthin is anintermediate in the abscisic acid biosynthetic pathway.

[0017] For the feed additive industry, xanthophyll marigolds areproduced primarily in Mexico, Peru, Africa, India, China and Thailand.Modern, commercial varieties include ‘Orangeade’, one of the originalxanthophyll producing varieties, and commercial improvements of‘Orangeade’, including ‘Deep Orangeade’ having larger flowers andgreater pigment yields, and ‘Scarletade’ an improvement for xanthophyllconcentration. Thus, ‘Orangeade’ is reported to contain xanthophylls atabout 9-12 mg/g of dry whole flower heads (including calyx), ‘DeepOrangeade’ is reported to have about 10-13 mg/g of those pigments, and‘Scarletade’ is said to contain about 12-18 mg/g of xanthophyll pigmentsin dry flower heads weighed with the calyx. These varieties areavailable from PanAmerican Seed Co., 622 Town Road, West Chicago, Ill.60185.

[0018] Whereas lutein is the major xanthophyll in marigold flowers, somecurrent varieties yield extract products with zeaxanthin ratios[zeaxanthin/(lutein+zeaxanthin)] typically in the 3 to 5 percent range(See Product Profile, Kemin Foods L.C., 600 E. Court Ave. Suite A, DesMoines, Iowa 50309). As is seen from the results hereinafter, zeaxanthinto lutein ratios obtained using ‘Scarletade’ are typically about 4 toabout 7 percent.

[0019] Moehs et al., Plant Mol. Biol., 45:281-293 (2001) analyzed thebiosynthesis of carotenoids in ornamental varieties of T. erecta,including a so-called wild type that had dark orange flowers, and plantswith yellow, pale yellow and white flowers. Among other findings, thoseworkers reported that although the different plants had a range inflower color from white to dark orange, the differences in those flowercolors were said to be due to the accumulation of very different amountsof the same carotenoid, lutein, rather than to accumulation of differentcarotenoid products or intermediates. The differences among the plantsstudied appeared to relate primarily to regulation of flux through thecarotenoid pathway, rather than to the specific type of carotenoidproduced or the accumulation of biosynthetic intermediates.

[0020] In addition, the so-called wild-type and mutant leaves werereported to contain about the same quantity of carotenoid pigments,regardless of flower color. Those pigments were different from thepigments present in the petals. Thus, the only pigment reported forpetals was lutein, whereas the leaves were reported to contain lutein aswell as β-carotene, violaxanthin and neoxanthin. As is seen from FIG. 1,β-carotene but not lutein can be a precursor to the latter two pigments.

[0021] The Moehs et al., authors also compared the T. erecta genes theyisolated with similar carotenoid-producing genes obtained from theleaves of Arabidopsis thaliana (Pogson et al., hereinafter). Identitiesbetween the gene products of about 70 to about 80 percent were reportedat the protein level, with a higher level if putative plastid targetingsignal peptides were excluded, and a lower level of identity at the DNAlevel. In leaves of A. thaliana, lutein is the predominant carotenoid,with β-carotene, violaxanthin and neoxanthin also being formed, but nozeaxanthin being normally accumulated.

[0022] Carotenoid biosynthesis in T. erecta is a complex systeminvolving many genes and possibly two pathways. The impact of geneticmutations on carotenoid production cannot be predicted a priori.However, classic breeding techniques have produced ‘Orangeade’, ‘DeepOrangeade’ and ‘Scarletade’ T. erecta variants that produce the elevatedlevels of xanthophylls noted above. These relatively recently bredavailable varieties have not been subject to treatments that inducegenetic mutations in an attempt to increase the zeaxanthin ratios.

[0023] Several workers have examined the effects of mutagens such asgamma irradiation, ethyl methanesulfonate (EMS) and nitrosomethylurea(NMU) on flowering plants, including marigolds. For example, Zaharia etal., Buletinul Institutului Agronomic Cluj-Napoca. Seria Agricultura44(1): 107-114 (1991) reported on the chlorophyll-deficient effects ofcarotenoids in the coleoptile after seeds of Zinnia elegans, Tageteserecta and Callistephus chinensis were irradiated with gamma irradiationin varying amounts. A paper by Geetha et al., Acta Botanica Indica,20(2):312-314 (1992) reports on the chlorophyll deficient effects ofgamma irradiation on Tagetes patula.

[0024] Diaconu, Agronomie, 34(1):17-21 (1991) reported on the effects ofEMS on germinating seeds from F₂ polycrosses of commonly-called potmarigolds, or Calendula, that are not even of the genus Tagetes. Thoseworkers noted a wide variation in flower color, inflorescence structure,yield and content of biologically-active substances in M₂-M₄ plants.

[0025] A study by Pogson et al., Plant Cell, 8:1627-1639 (1996) used EMSto mutagenize plants of Arabidopsis thaliana. This detailed study of4000 M₂ lines reported finding two loci in the carotenoid biosyntheticpathway in leaves that are involved with the production of lutein fromγ-carotene. Those loci were referred to as lut1 and lut2. The lut2 locuswas reported to be associated with the lycopene ε-ring cyclase enzyme,whereas the lut1 locus was reported to be associated with the lycopeneε-ring hydroxylase. Those workers noted (page 1631) that a decrease inlutein production was compensated for by an equimolar change in theabundance of other carotenoids, although only small amounts of thosechanges were due to an increased production of zeaxanthin.

[0026] Cetl et al., Folia Fac. Sci. Nat. Univ. Purkynianae Brun Biol.,21(1):5-56 (1980)reported extensive studies with T. erecta and otherTagetes species that from the meager descriptions appeared to all beornamental varieties. Among those studies, those authors examined theeffects of various concentrations of NMU on T. erecta seeds, andexamined more than about 2000 plants. All M₂ plants deviating from thephenotype of the parental cross were recorded, and M₃ plants from M₂seeds of the phenotypically different plants were studied.

[0027] Those workers assayed plant height, plant diameter, flower headdiameter and height of the flower head, as well as time to flowering,branching amount, branch length, cotyledon and leaf size, and flowerstalk length. No mention is made regarding flower color or carotenoidlevels in the leaves or petals.

[0028] Published PCT application WO 00/32788 of DellaPenna et al.asserts of a method of regulating carotenoid biosynthesis in marigolds.Those workers provide polynucleotide sequences said to be those thatencode the lycopene β-ring cyclase and lycopene β-ring hydroxylaseneeded for the preparation of zeaxanthin from lycopene. Also disclosedis a lycopene ε-ring cyclase useful along with the lycopene β-ringcyclase for the preparation of α-carotene from lycopene. No teaching ofthe lycopene ε-ring hydroxylase needed for lutein production isprovided.

[0029] Carotenoid biosynthesis is said to be regulated in PCTapplication WO 00/32788 by expression of a carotenoid synthesizingenzyme-encoding gene already present in marigolds such as those notedabove, or by use of an anti-sense RNA encoded by such a nucleotidesequence provided. No evidence of such regulation is provided in theapplication. The phenomenon known as co-suppression by which theaddition of a homologous gene causes both the native gene and transgenenot to be expressed is not dealt with by those workers. [See forexample, Fray et al., Plant Mol. Biol., 22:589-692 (1993) or Finnegan etal., Bio/Technology, 12:883-888 (September 1994).]

[0030] An increased ratio of zeaxanthin to lutein can also provide anattractive substrate for biotechnological production of additionalxanthophylls including the red xanthophyll, astaxanthin. Astaxanthin iswidely used as a pigmenting agent for cultured fishes and shellfishes.The complete biomedical properties of astaxanthin remain to beelucidated, but initial results suggest that it could play an importantrole in cancer and tumor prevention, as well as eliciting a positiveresponse from the immune system. [See Tanaka et al., Carcinogenesis15(1):15-19 (1994); Jyonouchi et al., Nutrition and Cancer 19(3):269-280(1993) and Jyonouchi et al., Nutrition and Cancer 16(2): 93-105 (1991).]

[0031] Astaxanthin supplied from biological sources, such ascrustaceans, yeast and green algae is limited by low yield and costlyextraction methods when compared with that obtained by organic syntheticmethods. Usual synthetic methods however, produce by-products that canbe considered unacceptable. It is therefore desirable to find arelatively inexpensive source of (3S, 3′S) astaxanthin to be used as afeed supplement in aquaculture and as a valuable chemical for otherindustrial uses.

[0032] One approach to increase the productivity of astaxanthinproduction in a biological system is to use genetic engineeringtechnology. Genes suitable for this conversion have been reported.

[0033] For example, Misawa et al. (See U.S. Pat. No. 6,150,130)specified DNA sequences including one isolated from the marine bacteriaAgrobacterium aurantiacus sp. nov. MK1 or Alcaligenes sp. PC-1 thatencodes a gene, referred to as crtW, used in the production ofastaxanthin from zeaxanthin as a substrate by way of 4-ketozeaxanthin.Kajiwara et al. (See U.S. Pat. No. 5,910,433) identified apolynucleotide molecule, referred to as bkt, isolated from Haematococcuspluvialis that encodes a polypeptide having a beta-C-4-oxygenaseactivity for the production of (3S, 3′S)astaxanthin from a hostmicroorganism or a plant. In addition, Hirschberg et al. (See U.S. Pat.No. 5,965,795) described another DNA gene sequence from Haematococcuspluvialis, referred to as crtO, that encodes an enzyme that synthesizesastaxanthin from zeaxanthin by way of 4-ketozeaxanthin. Still further,Cunningham (See WO 99/61652) reported isolation of a DNA that encodes aprotein having ketolase enzyme activity from Adonis aestivalis, a plantspecies having deep red flower color due in part to the accumulation ofthe ketocarotenoid astaxanthin.

[0034] It would therefore be useful if a marigold plant could beprovided whose flower petals or leaves or both contain a commerciallyuseful amount of xanthophylls and an altered ratio of lutein andzeaxanthin such that the usually reported 4 to about 7 percentzeaxanthin level were raised and the amount of lutein were decreased. Itwould also be useful if the ratios of other pigments could also beraised, and if such a plant had substantially the same phenotypicalcharacteristics as a usual marigold plant grown adjacent to it. Thepresent invention provides several such plants, flower petals, leaves,seed that produce them, hybrids, oleoresins, mixtures of zeaxanthin andlutein, and comestible materials containing zeaxanthin, lutein,α-cryptoxanthin, antheraxanthin, neoxanthin and violaxanthin, andβ-carotene dissolved or dispersed in a comestible medium.

BRIEF SUMMARY OF THE INVENTION

[0035] The present invention contemplates marigold plants whose petals,leaves or both flower petals and leaves contain one or more of anenhanced zeaxanthin ratio, an enhanced neoxanthin plus violaxanthinratio, an enhanced β-carotene ratio, an enhanced α-cryptoxanthin ratioan enhanced phytoene ratio or an enhanced phytofluene ratio compared tosuch a ratio present in a non-mutant marigold. In addition, the flowerpetals typically contain a measurable amount of zeta-carotene(ζ-carotene), whereas that compound is not measurable; i.e., is presentat less than 0.1 percent, in the petals of a non-mutant marigold plant.

[0036] A stated ratio is determined as a percentage of the first-namedpigment divided by the sum of the percentages of that pigment and luteinas determined by chromatographic techniques discussed hereinafter. Thus,the zeaxanthin ratio is illustratively defined herein aszeaxanthin/(zeaxanthin+lutein).

[0037] Preferably the petals, leaves or both the petals and leaves ofsuch a plant at least exhibits a zeaxanthin ratio greater than about1:10, preferably greater than about 2:10, up to about 1.0. That ratiocan be up to one because lutein cannot be detected in some leaves. Insome embodiments, the flower from which the petals are taken has axanthophyll content of about 4 to about 25 mg/g dry weight, whereas inother plants the petal xanthophyll content can be lower. The xanthophyllcontent of leaves is typically about 0.5 to about 1.25 mg/g dry weight.The flower petals and leaves are typically present in comminuted form.

[0038] The plant that produced the desired petals and leaves is a mutantwhose phenotype except as to carotenoids can be substantially the sameas that of an adjacently-grown non-mutant plant, or that phenotype canbe different. In one aspect, a contemplated marigold plant is a hybridbetween another contemplated mutant plant and a non-mutant in which thenon-mutant plant is a hybrid neither of whose parents are mutants.

[0039] Another aspect of the invention contemplates a marigold plant, ora regenerable portion thereof, whose flower petals or leaves or bothcontain one or more of an enhanced zeaxanthin ratio, an enhancedneoxanthin plus violaxanthin ratio, an enhanced β-carotene ratio, anenhanced α-cryptoxanthin ratio, an enhanced phytoene ratio or anenhanced phytofluene ratio and preferably at least a zeaxanthin ratiothat is greater than about 1:10, preferably greater than about 2:10, andup to about 1.0. The petals of a contemplated plant typically contain ameasurable amount of zeta-carotene, as discussed before.

[0040] A contemplated plant in one embodiment is a hybrid or latergeneration hybrid. A contemplated marigold plant of one aspect containsan amount of xanthophylls, measured as the saponified pigmentsextractable from the flowers that is about 4 to about 25 grams perkilogram of dry flowers or about 4 to about 25 mg/g dry weight. Acontemplated marigold of another aspect contains a lesser xanthophyllcontent. The pollen and an ovule of such a plant are separatelycontemplated. The regenerable portion of such a contemplated plantcomprises cells that include embryos, meristems, pollen, leaves,anthers, roots, root tips, and flowers, or protoplasts or callus derivedtherefrom.

[0041] Another embodiment contemplates a seed that on planting in asuitable environment and growth to maturity yields a marigold plantwhose flower petals or leaves or both contain one or more of an enhancedzeaxanthin ratio, an enhanced neoxanthin plus violaxanthin ratio, anenhanced β-carotene ratio, an enhanced α-cryptoxanthin ratio, anenhanced phytoene ratio or an enhanced phytofluene ratio and preferablyat least a zeaxanthin ratio that is greater than about 1:10, preferablygreater than about 2:10, and up to about 1.0. The petals of acontemplated plant again typically contain a measurable amount ofzeta-carotene, typically at least 1 percent or more, as discussedbefore.

[0042] A mutant marigold plant oleoresin having one or more of anenhanced zeaxanthin ratio, an enhanced neoxanthin plus violaxanthinratio, an enhanced β-carotene ratio, an enhanced α-cryptoxanthin ratio,an enhanced phytoene ratio or an enhanced phytofluene ratio relative toan oleoresin from a non-mutant marigold, and preferably at least azeaxanthin ratio that is greater than about 1:10, preferably greaterthan about 2:10, and up to about 1.0, is also contemplated. Acontemplated oleoresin also usually contains a measurable amount ofzeta-carotene, as discussed before.

[0043] A composition suitable for use as a food or feed supplement isalso contemplated. The food or feed supplement comprises a mixture ofzeaxanthin and lutein fatty acid esters dissolved or dispersed in acomestible medium, wherein the zeaxanthin ratio is greater than about1:10, preferably greater than about 2:10, and up to about 1.0. Anothercomposition suitable for use as a food or feed supplement compriseszeaxanthin and lutein dissolved or dispersed in a comestible medium,wherein the zeaxanthin ratio is greater than about 1:10, preferablygreater than about 2:10, and up to about 1.0.

BRIEF DESCRIPTION OF THE DRAWING

[0044] In the drawings forming a part of this disclosure,

[0045]FIG. 1 is a schematic representation of the biological synthesispathway for the production of lutein and zeaxanthin in plants in whichphytoene, the first C₄₀ carotenoid in the pathway, is converted inseveral steps (four arrows) to lycopene and then the γ-carotene thatcontains one β-ring, after which the pathway splits to form α-carotenethat contains one ε-ring and one β-ring or β-carotene that contains twoβ-rings, and after several steps, to lutein or zeaxanthin, respectively,and the zeaxanthin branch continuing to the epoxide-containingxanthophylls antheraxanthin, violaxanthin and neoxanthin.

[0046] As used herein, the term “zeaxanthin ratio” is defined as thequantity of zeaxanthin present in a dried flower petal or leaf dividedby the quantity of zeaxanthin plus lutein[zeaxanthin/(lutein+zeaxanthin)] present in that petal or leaf. The“neoxanthin plus violaxanthin ratio” is similarly calculated as theratio of neoxanthin+violaxanthin divided by the sum of those twopigments plus lutein. The “β-carotene ratio”, the “α-cryptoxanthinratio”, the “phytoene ratio” and the “phytofluene ratio” are similarlycalculated using the appropriate pigment amount as the numerator and thesum of either pigment plus lutein as the denominator. Those pigmentquantities are determined by high performance liquid chromatography(HPLC) after saponification of a dried flower petal or leaf extract asdiscussed hereinafter so that the amount of each of lutein andzeaxanthin (or other pigment) is measured in the free compound form,e.g., alcohol form for lutein and zeaxanthin, present aftersaponification rather than in the esterified form that is present in thefresh flower petal, and chlorophyll that may be present in a leafextract is destroyed.

[0047] The word “oleoresin” is used herein to mean an extract of planttissues that contains plant pigments such as the xanthophylls discussedherein in their esterified forms, sometimes accompanied by amounts ofother plant products and pigments such as other carotenoids such asβ-carotene, as well as small amounts of solvent such as hexane oracetone, typically less than 1 percent organic solvent. Xanthophylls aretypically present as mono- or diesters in flower petals and aretypically present as free alcohols in marigold leaves. Carotenes such asβ-carotene or lycopene are present as free, non-chemically-combinedcompounds. Chlorophyll is present in marigold leaves and largely absentin the petals. Thus, an oleoresin prepared from flower petals containsxanthophyll esters and is largely free of chlorophyll, whereas anoleoresin prepared from marigold leaves contains chlorophyll and freexanthophylls. Both chlorophyll and xanthophyll esters are decomposed bysaponification of the oleoresin. A contemplated oleoresin is a solid orsemi-solid material.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention contemplates marigold plants, seeds, flowerpetals, leaves and materials that can be prepared therefrom. Acontemplated plant additionally has flower petals, leaves or both thatcontain an enhanced carotenoid ratio as compared to previously knownmarigold plants. The petals and/or leaves of a contemplated plant thuscontain one or more of an enhanced zeaxanthin ratio, an enhancedneoxanthin plus violaxanthin ratio, an enhanced β-carotene ratio, anenhanced α-cryptoxanthin ratio, an enhanced phytoene ratio or anenhanced phytofluene ratio. The leaves of a contemplated plant can befree of lutein. These contemplated marigold plants are T. erecta, ascompared to T. patula or T. tenuifolia, or other Tagetes species. Inaddition, a contemplated plant can be a xanthophyll marigold, as suchplants have been described before and are understood by workers of skillin this art.

[0049] The usual ratio of zeaxanthin to zeaxanthin+lutein in marigoldpetals is on the order of about 1:15 to about 1:25, so that when onlyzeaxanthin and lutein amounts are used for calculations, zeaxanthin isabout 5 to about 7 percent of the amount of lutein plus zeaxanthin. Anarticle by Quackenbush et al., J. Assoc. Off. Agri. Chem., 55:617-621(1972) reported a zeaxanthin to lutein ratio in one group of Americanyellow T. erecta marigold flower petals that was unusually high at about1:4.4, whereas the total concentration of xanthophylls in those petalswas unusually low at about 0.4 mg/g dry weight. A Mexican variety wassaid by those authors to contain 11.1 percent zeaxanthin whenlyophilized petals were assayed and 3.8 percent when fresh petals wereassayed. The higher value is not in keeping with the remainder of thedata and is believed to be incorrect. The preferred zeaxanthin ratio inpetals contemplated here is even larger, being greater than about 1:10and preferably greater than about 2:10, as will be discussedhereinbelow, and the amount of petal xanthophylls is preferably at leastabout 4 mg/g dry weight.

[0050] A contemplated marigold plant has flower petals that contain azeaxanthin ratio greater than about 1:10 and preferably greater thanabout 2:10. More preferably still, a contemplated marigold plant hasflower petals that contain a zeaxanthin ratio greater than about 3:10.Most preferably, that ratio is greater than 5:10, and can be about 1.0.The flower from which the petals are taken has a xanthophyll content ofabout 4 to about 25 mg/g dry weight, and preferably about 10 to about 20mg/g dry weight. Such a marigold plant also preferably has leaves thatcontain a zeaxanthin ratio greater than about 1:10 and preferablygreater than about 2:10. More preferably still, a contemplated marigoldplant has leaves that contain a zeaxanthin ratio greater than about3:10. Most preferably, that ratio is greater than 5:10, and can be about1.0. The contemplated leaves have a xanthophyll content of about 0.2 toabout 1.25 mg/g dry weight, and preferably about 0.5 to about 1 mg/g dryweight.

[0051] In some embodiments, the lutein concentration of the petals of acontemplated plant contain about 80 to about 90 percent of the luteinpresent in a parental, non-mutant plant. In other embodiments, theamount of lutein is less than about 75 percent of that present in anon-mutant plant. In still further embodiments, the amount of luteinpresent in the flower petals is less than about 15 percent of thatpresent in the petals of a non-mutant marigold plant.

[0052] A contemplated marigold can also exhibit differences in ratios ofone or more other pigments relative to lutein. Thus, the neoxanthin plusviolaxanthin ratio in a parental plant can be about 0.01 to about 0.022for a non-mutant petal extract and about 0.17 to about 0.33 in leaves.That ratio in contemplated mutant plant petals and leaves of onepreferred embodiment is about 1:5 (0.2) to about 1:1 (1). Neoxanthin andviolaxanthin are measured together as they are difficult to separatechromatographically.

[0053] The β-carotene ratio in non-mutant plants is typically about lessthan 0.007 for flower petals and about 0.3 to about 0.4 for leaves. Thatratio is about 0.05 to about 0.9 for flower petals and about one forleaves of mutant marigold plants.

[0054] α-Cryptoxanthin typically constitutes less than one percent ofcolored carotenoids of non-mutant plant petals and the α-cryptoxanthinratio is consequently about 0.01 in non-mutant flower petals. Theα-cryptoxanthin ratio is about 0.25 to about 0.9 in the petals of somepreferred mutated plants.

[0055] Phytoene can be present in petals at about 3 to about 0.3 percentof the carotenoids in non-mutant plants and can be present in at about35 percent in some mutant flower petals that typically contain a reducedamount of lutein. Exemplary phytoene ratios can be from about 0.3 toabout 1 in a contemplated plant as compared to phytoene ratios of about0.003 to about 0.03 in non-mutant plants. Phytoene concentrations arelargely unchanged in leaves of mutant plants as compared to non-mutantplant leaves.

[0056] Phytofluene amounts in non-mutant plant petals are typicallyabout the same as those observed for phytoene, whereas the amountpresent in the petals of a contemplated mutant plant is generally about40 to about 70 percent of the phytoene amount. The phytofluene ratio fora non-mutant plant is usually about 0.005 to about 0.03, whereas thatratio for a contemplated mutant plant is about 0.2 to about 1.Phytofluene has not been observed in leaf extracts.

[0057] The enhancements observed in the above ratios are typically atleast about two-fold. In particular embodiments, a ratio can be enhancedby about ten- to about one hundred-fold.

[0058] The petals or leaves or both of a particular plant can have oneor more of the above-recited enhanced ratios. In usual practice, two ormore of the before-described enhanced ratios are present. Thus, forexample, the zeaxanthin ratio and the β-carotene ratio can be enhanced,or the zeaxanthin ratio and the neoxanthin plus violaxanthin ratio canbe enhanced. Similarly, three or more of the ratios can be elevated.

[0059] α-Cryptoxanthin is a particularly interesting compound in that ithas been found to be present in relatively high levels in a number ofmutants and is not otherwise readily available. Thus, α-cryptoxanthinwas present at about 20 to about 40 percent of colored carotenoids insome mutants. The α-cryptoxanthin ratio of such plants was consequentlygreatly enhanced as compared to those plants whose petals contained moreusual amounts of carotenoids.

[0060] The petals of a contemplated plant typically contain a measurableamount of zeta-carotene (ζ-carotene), whereas that pigment is notpresent in a measurable amount; i.e., present at less than 0.1 percent,in the petals of a non-mutant marigold plant. Typically, zeta-caroteneis present in an amount of at least about 1 percent of the petalcarotenoids. That amount of zeta-carotene can be in the range of about 3to about 7 percent in some embodiments, and at about 20 percent in otherembodiments.

[0061] As already noted, xanthophylls such as lutein and zeaxanthin arepresent in flower petals primarily as mono- or diesters of fatty acidssuch as lauric, myristic, palmitic, stearic, oleic or the like, ratherthan as free compounds. As such, when a zeaxanthin or other xanthophyllratio is discussed herein, that ratio is determined by extracting one ormore flower petals with hexane or other appropriate solvent to obtain acomposition such as an oleoresin comprised of esterified xanthophylls.That composition is then saponified using a base such as potassiumhydroxide to cleave the esters and form free carotenoid alcohols. Thefree carotenoid xanthophyll alcohols are thereafter separated from thesaponification reaction mixture and separated as desired using highperformance liquid chromatography (HPLC). The ratios of materialspresent are determined by the areas under the appropriate HPLC peaksusing standard methods of integration.

[0062] The analytical method utilized herein to determine the pigmentratios is exemplified hereinafter, and provides similar results to thosepublished by others, with different specific techniques being used bydifferent laboratories largely for reasons of convenience. Using theprocedure preferred here, flowers approximately 98 percent fully openedare selected for analysis. Petals are removed about one-third of thedistance from the flower center from the selected flowers.

[0063] Leaves can be harvested and extracted at substantially any time.Xanthophylls are typically present as free compounds in leaves as arecarotenes. Chlorophyll present in leaves is also extracted with thecarotenoid pigments so assays are carried out after saponification ofthe extract as that treatment destroys chlorophyll. Leaves are assayedfor carotenoid content as are the petals.

[0064] A standard analytical method used in the industry for determiningcarotenoid levels in plant extracts is that of the AOAC 1984, OfficialMethods of Analysis (14^(th) ed), the Association of Official AnalyticalChemists, Arlington, Va., USA, the results of whose assays are similarto those obtained herein.

[0065] A contemplated marigold plant is a mutant of a parental line.That is, a first line or cross or seed is treated with a mutagen(mutagenized) to provide a mutagenized plant that is typicallyself-pollinated (selfed) one or more times. A plant contemplated hereincan arise from the mutagenesis itself, from one of the selfings or froma cross of a mutagenized plant or offspring with another mutagenized ornon-mutagenized plant.

[0066] Substantially any kind of mutagen can be used to produce acontemplated plant, and exemplary mutagens are discussed hereinafter.Although some contemplated mutant marigolds have a phenotype that issubstantially different from that of adjacently-grown non-mutantmarigold parental plant, other contemplated mutants exhibitsubstantially the same phenotype as that of an adjacently-grownnon-mutant parental plant, except for phenotypic traits related tocarotenoids. More specifically for the latter plants, when one comparesplant properties such as plant height, plant diameter, flower headdiameter, flower head height, time to flowering, branching amount,length of branches, flower stalk length, hypocotyl length, cotyledonlength and cotyledon width between a parent and a mutant plant, thevalues of those properties for some contemplated mutant plants are eachwithin about 90 percent of those of the parental plant, including thestandard deviations in the measurements. More preferably, the values forthose properties of the mutant are within about 95 percent of theparent, and most preferably, the values are the same, within thestandard deviation. On the other hand, other mutant plants differgreatly in one or more phenotypic traits.

[0067] A carotenoid-related phenotypic difference between the parentaland mutant plants is the quantity of xanthophyll pigment that can beobtained from the flowers of the mutant. Parental plants such as‘Scarletade’ or ‘Deep Orangeade’ typically have about 10 to about 18mg/g dry whole flower head weight of extractable xanthophyll pigments. Acontemplated mutant plant preferably contains about the same amount ofcarotenoid in the flower petals, but can contain as little as about 4mg/g dry weight, particularly where the ratio of zeaxanthin to lutein isvery high such as about 9:1 or greater.

[0068] The leaves of a contemplated marigold can also exhibit aphenotypic difference between the parental and mutant plants as to thecarotenoid content as well as one or more of the before-discussedcarotenoid ratios present in the leaves as measured in a saponifiedoleoresin. The previously noted paper of Moehs et al., Plant Mol. Biol.,45:281-293 (2001) reported that leaf carotenoid ratios and contents wereconstant, whereas carotenoid concentration in the petals differed. Here,it is found that one or more of the before-mentioned zeaxanthin ratio,antheraxanthin ratio, neoxanthin plus violaxanthin ratio, phytoeneratio, phytofluene ratio, β-carotene ratio and α-cryptoxanthin ratio inmutant plants differed considerably from parental non-mutant plants. Inaddition, the petals of each of the mutant plants examined exhibited ameasurable amount of zeta-carotene, whereas no measurable amountzeta-carotene was observed to be present in the parental non-mutantplants.

[0069] Phenotypic comparisons are made between adjacently-grown plants.As used herein, the term “adjacently-grown” is used to mean plants grownunder as similar conditions of light, heat, growth medium, humidity andnutrients as can be achieved so that growth conditions do not govern thephenotype. For greenhouse-grown plants, “adjacently-grown” means plantsgrown under conditions as similar as possible on the same bench. Forfield-grown plants, “adjacently-grown” means plants grown underconditions as similar as possible in the same or adjoining fields.

[0070] Mutagenic agents useful for altering plants are well known in theart, as are methods of using such agents. Exemplary chemical mutagensinclude nitrosomethylurea (NMU), ethyl methanesulfonate (EMS), methylmethanesulfonate, diethyl sulfate, nitrosoguanidine, andethylnitrosourea of which EMS is preferred herein. NMU can be used asdiscussed in Cetl et al., Folia Fac. Sci. Nat. Univ. Purkynianae Brun.Biol., 21(1): 5-56 (1980), whereas EMS is typically utilized at about0.25 to about 1 percent by volume (v/v), and preferably at about 0.2 toabout 0.8 percent. Gamma irradiation is also a useful mutagenic agentwhen used to irradiate seeds at a dose of 200 to about 20,000 rads (0.2to about 20 krads).

[0071] Regardless of the mutagen used, the phenotype of the resultingmutant plant, including carotenoid-related traits such as the zeaxanthinratio and the amount of xanthophylls in the petals, is usuallysubstantially identical to that of the parent, so that a very largepercentage of the mutants obtained are not useful. In addition, plantsseeming to have the same phenotype as the parent need to be screened tolocate a desired mutant plant. Those screenings, although tedious, areroutinely carried out and involve analysis of carotenoid pigments fromone or more single flower petals or leaves or both. Thus, thepreparation of a desired mutant is a relatively rare, but repeatableevent. For example, in one study herein, only twenty-three usefulmutants were obtained from almost 22,000 mutant plants examined. Inanother study, about twenty-four useful mutants out of about 8,200examined plants were obtained.

[0072] As already noted, a contemplated plant can be a plant that growsfrom the mutagenized seed or can be a selfing or cross. In one preferredembodiment, a contemplated marigold is a hybrid formed by crossing theflowers of two plants that arose from two different mutagenized plantsfrom independent M₁ plants (M₂×M₂). In another embodiment, acontemplated marigold is a hybrid formed by crossing the flowers of oneplant that arose from one mutagenized plant with a non-mutagenizedplant. In still another embodiment, a contemplated plant is a hybridformed by back-crossing a hybrid with one or the other of its immediateparental flowers. The product of the crossing of two different hybridplants is contemplated as is the product of the selfing of a hybrid.

[0073] The present invention also contemplates the pollen and an ovuleof a contemplated plant. The regenerable portion of a contemplated plantis also itself contemplated and includes cells selected from the groupconsisting of embryos, meristems, pollen, leaves, anthers, roots, roottips, and flowers, or protoplasts or callus derived therefrom. Methodsfor regenerating plants from cells are well known to those skilled inthe art, and dicotyledonous plants such as marigolds are particularlyamenable to such regeneration.

[0074] A marigold oleoresin comprised of fatty acid esters of lutein andzeaxanthin in which the zeaxanthin ratio is greater than about 1:10 andpreferably greater than about 2:10 is also contemplated. Morepreferably, that ratio is greater than about 3:10 and is most preferablyabout 1.0. A contemplated marigold oleoresin contains a zeaxanthin ratioas is present in the petals or leaves of a contemplated marigold asdiscussed before. Oleoresins are items of commerce and are sold toprocessers for further treatment in the production of human or otheranimal food or feed supplements. A contemplated oleoresin also typicallycontains a measurable amount of zeta-carotene.

[0075] In an illustrative marigold oleoresin preparation, xanthophyllesters; i.e., zeaxanthin or mixture of zeaxanthin and lutein esters andpossibly other xanthophyll esters and carotenes such as zeta-carotene,are extracted from dried marigold flowers with hexane, acetone, ethylacetate, toluene, tetrahydrofuran (THF) and the like organic solvent, ora mixture thereof. The extraction is carried out according to proceduresknown in the art. The solvent(s) is removed, resulting in an extractthat typically contains a high level of the xanthophyll esters and isabout 99 percent and preferably about 99.9 percent free of theextracting organic solvent; i.e., contains less than about 1 percent andpreferably less than about 0.1 percent organic solvent by weight. Theresulting solvent-free extract is referred to as a marigold oleoresin. Aleaf extract is similarly prepared, and contains free xanthophylls,carotenes and chlorophyll.

[0076] A composition suitable for use as a food or feed supplement forhuman or other animals such as poultry like chickens and turkeys, fishlike trout and salmon and crustaceans like shrimp, lobsters and crabs isalso contemplated. A contemplated food or feed supplement can be used toprovide color to the skin and fat of those animals as well as to the eggyolks of such animals, and particularly chickens.

[0077] One food or feed supplement comprises a mixture of fatty acidesters of zeaxanthin alone or zeaxanthin, lutein and other carotenoidsas are present in a marigold oleoresin. That mixture of mostly fattyacid esters is dissolved or dispersed in a comestible medium, whereinthe zeaxanthin and lutein fatty acid esters are present at a zeaxanthinratio that is greater than about 1:10, preferably greater than about2:10, more preferably greater than about 3:10, and up to about 1.0. Thisfood or feed supplement can thus be prepared by suitable purification ofa before-described oleoresin as by dissolution and filtration, followedby dissolution or dispersion of the purified mixed esters in anappropriate comestible medium.

[0078] In some embodiments, the comestible medium is an edibletriglyceride oil, whereas in other embodiments the comestible medium isa binding agent such as is frequently found in pharmaceutical productssuch as pills and tablets (a pharmaceutically acceptable binding agent).For tablets or capsules, the xanthophyll ester content of the admixturemeasured as free xanthophyll is typically about 0.1 to about 25milligrams per tablet or capsule, and more usually about 5 to about 20milligrams per tablet or capsule.

[0079] Binding agents and adhesives preferably impart sufficientcohesion to solids to permit normal processing such as sizing,lubrication, compression and packaging, but still permit a tablet orcapsule to disintegrate and the composition to dissolve upon ingestion.Exemplary binding agents include lactose monohydrate, acacia,tragacanth, sucrose, gelatin, glucose, cellulose or saccharide materialssuch as, but not limited to, microcrystalline cellulose, croscarmellosesodium, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose(Klucel™), ethyl cellulose (Ethocel™), methyl cellulose and sodiumcarboxymethyl cellulose (e.g., Tylose™), pregelatinized starch (such asNational™ 1511 and Starch 1500), polysaccharide acids, alginic acid andsalts of alginic acid, magnesium aluminum silicate, polyethylene glycol,guar gum, bentonites, polyvinylpyrrolidone (povidone), andpolymethacrylates.

[0080] Exemplary edible oils include candelilia, coconut, cod liver,cotton seed, menhaden, olive, palm, corn, soybean, peanut, poppy seed,safflower and sunflower oil. The use of an oil having a relatively highconcentration of unsaturated fatty acids is preferred; i.e., the use ofan oil having an iodine value of about 100-150 is preferred. Theadmixture is typically carried out using a high shear mixing apparatus,as is well known. Co-solvents and additives such as ethanol andα-tocopherol, respectively, can also be present as is noted in U.S. Pat.No. 5,382,714.

[0081] In another embodiment, the mixture of zeaxanthin and lutein,zeaxanthin alone, or other carotenoid mixture is provided in the form ofgenerally spherical small pellets containing 0.5 to about 20 percent,and preferably about 1 to about 4 percent, of the xanthophyll that areconventionally referred to as “beadlets”. These beadlets can be usedadmixed in a desired amount into human food such as ready to eat cerealsas is disclosed in U.S. Pat. No. 5,270,063 or admixed into chicken orother animal feed as are the beadlets or other particles disclosed forthe feed additive in U.S. Pat. No. 5,849,345, U.S. Pat. No. 5,695,794,U.S. Pat. No. 5,605,699 and U.S. Pat. No. 5,043,170.

[0082] Exemplary beadlets are water-insoluble and are prepared byencapsulation of a xanthophyll composition by cross-linked gelatin or analginate such as sodium alginate as is disclosed in U.S. Pat. No.4,670,247. A water insoluble beadlet containing the desiredcarotenoid(s) is prepared by forming an emulsion containing thecarotenoid(s), water, gelatin, and a sugar. The emulsion is convertedinto droplets that are individually collected in a mass of starchypowder in such a manner that the particles from the droplets are keptseparated from each other until their particulate form is permanentlyestablished. The carotenoid-containing particles are separated from thestarchy collecting powder, and heat-treated at a temperature of about90° C. to about 180° C. The heat treatment step insolubilizes thegelatin matrix of the beadlet by a reaction between the carbonyl groupof the sugar with the free amino moieties of the gelatin molecule. Theresulting beadlets are water-insoluble and exhibit increased stabilityto the stresses of feed pelleting. The cross-linking process utilizesthe ingredients employed in making the beadlet and does not requireaddition of a cross-linking reagent or additive to the composition.

[0083] U.S. Pat. No. 5,695,794 discloses another form of beadlets thatcan be adapted for use herein as an additive for poultry feed. Thus,beadlets having diameters of about 30 to about 55 microns are preparedby spraying a molten solution of a desired amount of carotenoid(s);i.e., zeaxanthin, a mixture of zeaxanthin and lutein, or othercarotenoid mixture described herein, in hydrogenated vegetable oil suchas hydrogenated cotton seed oil, wheat-germ oil, safflower oil, soybeanoil and the like, that also can contain mono- and diglycerides such asthose prepared from hydrogenated soybean mono- and diglycerides,cottonseed mono- and diglycerides and the like, as well as citric acidand 2,6-di-tert-butyl-4-methylphenol (BHT) as antioxidants. Otherantioxidants such as ethoxiquin, vitamin E and the like can also beused, as is well known. The molten mixture is sprayed at a temperatureof about 160° F. (about 70° C.) into a cyclonic airstream of a spraychiller such as available from Niro, Inc., Columbia, Md. to produce thebeadlets that solidify on cooling. The cooled beadlets are dusted withan anticaking agent such as fumed silica, calcium phosphate, powderedstarch or cellulose as are well known to form the beadlets that arepreferably added to the feed as supplement. An exemplary beadletcontains about 10 to about 100 milligrams of zeaxanthin per gram (mg/g)and preferably at about 10 to about 50 mg/g.

[0084] Animal feeds to which a contemplated zeaxanthin orzeaxanthin-lutein mixture are added are well known in the art. Theabove-noted U.S. Pat. No. 5,849,345, U.S. Pat. No. 5,695,794, U.S. Pat.No. 5,605,699 and U.S. Pat. No. 5,043,170 provide exemplary diets thatare particularly useful for poultry. U.S. Pat. No. 5,935,624 and U.S.Pat. No. 2,918,370 provide further illustrative poultry diets.

[0085] U.S. Pat. No. 5,258,189 teaches the addition of β-carotene to aready to eat cereal product for humans in which the β-carotene isadmixed with a cooked cereal product dispersed in a vegetable oil or indry form. Zeaxanthin or a mixture of zeaxanthin and lutein as discussedelsewhere herein can be used at a desired level in place of β-carotenein a similar food product.

[0086] Another composition suitable for use as a food or feed supplementcomprises a mixture of zeaxanthin and lutein dissolved or dispersed in acomestible medium, wherein the zeaxanthin ratio present is at a greaterthan about 1:10, preferably greater than about 2:10, and up to about1.0. This composition contains saponified xanthophylls that are freezeaxanthin and lutein as compared to the esters that are present in amarigold oleoresin.

[0087] Methods are well known for saponifiying marigold oleoresins toprovide free xanthophylls. See, for example, Tyczkowski et al., PoultrySci. 70(3): 651-654, 1991; and U.S. Pat. No. 5,382,714, that lutein wascrystallized from the saponified marigold oleoresin by the addition oforganic solvents.

[0088] In addition, Ausich et al. U.S. Pat. No. 5,648,564 teaches theproduction of crystalline lutein from a marigold oleoresin by admixingthe oleoresin with a composition containing propylene glycol and anaqueous alkali, preferably potassium hydroxide, to form a reactionmixture of which oleoresin and propylene glycol together constitute atleast 75 weight percent. The reaction mixture so formed is maintained ata temperature of about 65° C. to about 80° C. for a time period(typically at least 3 hours) sufficient to saponify the xanthophyllester and form a saponified reaction mixture that contains freexanthophyll in the form of crystals. The saponified extract is admixedwith a diluting amount of water to dissolve the water-soluble impuritiesand reduce the viscosity of the reaction mixture. The diluted admixtureis gently admixed until homogeneous and then filtered to collect thexanthophyll crystals. The collected xanthophyll crystals are washed withwarm water, and dried. No organic solvent other than propylene glycol isused in the isolation and purification of the xanthophyll from thexanthophyll ester-containing oleoresin. The dried xanthophyll crystalsso formed are typically admixed with a comestible medium such as thetriglyceride discussed above. The xanthophyll content of the admixtureis typically about 0.1 to about 35 percent by weight, and preferablyabout one to about ten percent by weight.

[0089] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 EMS Treatment of Tagetes erecta ‘Scarletade’

[0090] Seeds of Tagetes erecta xanthophyll marigold denominated‘Scarletade’ (commercially available from PanAmerican Seed Co. 622 TownRoad, West Chicago, Ill. 60185) were treated with ethyl methanesulfonate(EMS, commercially available from Sigma Chemical Co., St. Louis, Mo.63178). Approximately 2,500 seeds were added to 400 ml of 0.4% (v/v) or0.8% (v/v) EMS and were stirred gently for eight hours at ambienttemperature. During a four-hour period following the EMS treatment, theseeds were washed sixteen times, each wash using continuous stirringwith 400 ml distilled water. The treated seeds, identified as M₁ seeds,were then sown in trays containing soilless potting mix.

[0091] After several weeks, the seedlings were transplanted into potscontaining soilless potting mix and maintained in the greenhouse.Flowers produced by those plants were naturally self-pollinated. Theresulting seeds, identified as M₂ seeds, were harvested fromapproximately 2,300 plants. Of these 2,300 plants, approximately 1,500were grown from seeds treated with 0.4% EMS and approximately 800 weregrown from seeds treated with 0.8% EMS. To facilitate identification ofmutant plants, the M₂ seeds from each of 50 M₁ plants were combined intoone lot, resulting in a total of 47 seed lots. During the summer of theyear 2000, 500 seeds from each of the 47 lots were sown and theresulting plants were field-grown at PanAmerican Seed Co. in SantaPaula, Calif. 93060.

EXAMPLE 2 HPLC Screening of EMS-Treated Tagetes erecta ‘Scarletade’

[0092] EMS-treated ‘Scarletade’ plants were field-grown at PanAmericanSeed Co. in Santa Paula, Calif. 93060, and were screened by HPLC foraltered zeaxanthin ratio. Flowers approximately 98% fully opened wereselected for analysis. From each flower, one petal was removed one-thirdof the distance from the flower center and placed in a 3.5″×0.75″ glassvial containing approximately 5 grams of glass beads. Vials werepackaged with dry ice until stored at −80° C.

[0093] For analysis, solvent delivery and aliquot removal wereaccomplished with a robotic system comprising a single injector valveGilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000 W. BeltlineHighway, Middleton, Wis.]. For saponification, 3 ml of 50% potassiumhydroxide hydro-ethanolic solution (4 water:l ethanol) was added to eachvial, followed by the addition of 3 ml of octanol. The saponificationtreatment was conducted at room temperature with vials maintained on anIKA HS 501 horizontal shaker [Labworld-online, Inc. Wilmington, N.C.]for fifteen hours at 250 movements/minute, followed by a stationaryphase of approximately one hour.

[0094] Following saponification, the supernatant was diluted with 0.9 mlof methanol. The addition of methanol was conducted under pressure toensure sample homogeneity. Using a 0.25 ml syringe, a 0.1 ml aliquot wasremoved and transferred to HPLC vials for analysis.

[0095] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete with aquaternary pump, vacuum degassing system, six-way injection valve,temperature regulated autosampler, column oven and Photodiode Arraydetector was used [Agilent Technologies available through UltraScientific Inc., 250 Smith Street, North Kingstown, RI.]. The column wasa Waters YMC 30, 5-micron, 4.6×250 mm with a guard column of the samematerial [Waters, 34 Maple Street, Milford, Mass.]. The solvents for themobile phase were 81 methanol: 4 water: 15 tetrahydrofuran (THF)stabilized with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injectionswere 20 μl. Separation was isocratic at 30° C. with a flow rate of 1.7ml/minute. The peak responses were measured by absorbance at 447 nm.

[0096] Using this protocol, the results from the first 2,546 sampleswere statistically analyzed to establish average values for lutein andzeaxanthin content. Because this was a semi-quantitative analyticalscreen, peak area values were used. To identify a mutant having a higherthan average lutein and/or zeaxanthin concentration, a value of threestandard deviations greater than the average was calculated. Thecalculated peak area means, standard deviations and zeaxanthin ratiosare shown in Table 1, below. TABLE 1 Lutein and Zeaxanthin ConfidenceInterval Calculations Peak Area Peak Area Statistic Lutein ZeaxanthinRatio (%) Mean 775.0 41.6 5.03 Standard 263.2 16.4 0.71 deviation (sd)Mean + 3 sd 1564.6 90.9 7.16

[0097] Based on the above values, samples were selected having luteinpeak areas greater than 1565 and/or zeaxanthin peak areas greater than91. Samples were also selected only for high lutein peak area, and forzeaxanthin ratios greater than 10 percent. A total of 88 mutants wereidentified from 21,754 assayed samples using these selection parameters.The total number of mutants resulting from each EMS seed treatment isshown in Table 2, below. TABLE 2 Correlation of ‘Scarletade’ Mutants toEMS Treatment Selection 0.4% EMS 0.8% EMS Total Parameter TreatmentTreatment Plants Zeaxanthin 10 13 23 Ratio > 10% Lutein > 1566 and 18 1028 Zeaxanthin > 91 Lutein > 1566 and 20 7 27 Zeaxanthin < 91 Lutein <1566 and 7 3 10 Zeaxanthin > 91

[0098] More specific results of those assays as to relative levels oflutein and zeaxanthin are shown in Table 3, below. TABLE 3 Identified‘Scarletade’ Mutants Plant Lutein Zeaxanthin Percent Percent IdentifierArea Area Zeaxanthin EMS Used 124-257 2.115 55.635 96.34 0.4 119-4949.254 131.036 93.40 0.8 112-263 8.095 35.273 81.33 0.4 118-036 11.44131.691 73.47 0.8 088-452 2.94 6.689 69.47 0.4 118-035 11.289 23.95167.97 0.8 114-334 58.24 97.968 62.72 0.4 117-185 39.002 44.027 53.03 0.8108-108 13.424 10.155 43.07 0.4 088-425 8.959 4.394 32.91 0.4 094-2387.285 3.063 29.60 0.4 110-308 46.753 14.248 23.36 0.4 132-346 31.0368.856 22.20 0.8 100-334 282.987 54.298 16.10 0.8 101-331 246.402 46.46715.87 0.8 100-198 119.381 21.449 15.23 0.8 101-190 139.027 23.125 14.260.8 114-315 351.524 56.898 13.93 0.4 100-470 189.703 27.743 12.76 0.8117-348 369.903 43.315 10.48 0.8 132-266 374.096 43.8 10.48 0.8 123-31060.743 6.818 10.09 0.4 116-106 453.538 50.287 9.98 0.8

[0099] About 21,700 plants exhibited typical zeaxanthin ratios of about4 to about 7 percent (about 1:25 to about 1:15). The above dataillustrate the relative rarity of the mutations contemplated, as well asthe almost equal number of plants that exhibit reduced zeaxanthinlevels. The data also do not show a preference for the use of one levelof mutagen versus the other used here.

EXAMPLE 3 EMS Treatment of Tagetes erecta 13819

[0100] Seeds of Tagetes erecta xanthophyll marigold named 13819(aproprietary breeding selection of PanAmerican Seed Co. 622 Town Road,West Chicago, Ill. 60185) were treated with ethyl methanesulfonate (EMS,commercially available from Sigma Chemical Co. St. Louis, Mo. 63178).Approximately, 7,000 seeds were added to 600 ml of 0.2% (v/v) or 0.4%(v/v) EMS and stirred gently for eight hours at ambient temperature.During a four-hour period following the EMS treatment, the seeds werewashed sixteen times, each wash using continuous stirring with 600 mldistilled water.

[0101] The treated seeds, identified as M₁ seeds, were then sown intrays containing soilless potting mix. After three to four weeks, theseedlings were transplanted into the field. Flowers produced by theseplants were bagged to prevent cross-pollination, and were permitted tospontaneously self-pollinate. The resulting seeds, identified as M₂seeds, were harvested from approximately 2,391 plants. Of these plants,approximately 951 were grown from seeds treated with 0.2% EMS andapproximately 1,440 were grown from seeds treated with 0.4% EMS.

[0102] To facilitate identification of mutant plants, the M₂ seeds fromeach of 50 plants were combined into one lot. This grouping resulted ina total of 48 seed lots. From late October through mid-November of theyear 2000, 1000 seeds from each of 15 lots of the 0.4% EMS treatmentwere sown and 700 plants of each lot were greenhouse-grown at SeaviewNursery in El Rio, Calif. 93060. In addition, 1,500 seeds from all ofthe 48 lots were sown in late October of the year 2000, and 765 plantsfrom each of the lots were field-grown at Semillas Pan American ChileLTDA, in Pichidegua, Chile.

EXAMPLE 4 HPLC Screening of EMS-Treated Tagetes erecta 13819

[0103] EMS-treated 13819 M₂ plants were greenhouse-grown at SeaviewNursery in El Rio, Calif. 93060 and field-grown at Semillas PanAmericanChile LTDA, in Pichidegua, Chile, and were screened for alteredzeaxanthin ratio. Flowers approximately 98% fully opened were selectedfor analysis. From these flowers, petals were removed one-third of thedistance from the flower center. Approximately 100 mg of petal tissuewas placed in plastic bags and stored frozen until analysis. Dry weightwas determined for two petals that were placed in 3.5″×0.75″ glass vialscontaining approximately 5 grams of glass beads.

[0104] For analysis, solvent delivery and aliquot removal wereaccomplished with a robotic system comprising a single injector valveGilson 232XL and a 402 2SlV diluter. For saponification, 3 ml of 50%potassium hydroxide hydro-ethanolic solution (4 water: 1 ethanol) wasadded to each vial, followed by the addition of 3 ml octanol. Thesaponification treatment was conducted at room temperature with vialsmaintained on an IKA HS 501 horizontal shaker for fifteen hours at 250movements per minute followed by a stationary phase of approximately onehour.

[0105] Following saponification, the supernatant was diluted with 0.9 mlof methanol. The addition of methanol was conducted under pressure toensure sample homogeneity. Using a 0.25 ml syringe, a 0.1 ml aliquot wasremoved and transferred to HPLC vials for analysis.

[0106] For HPLC analysis, a Hewlett Packard 1100 complete with aquaternary pump, vacuum degassing system, six-way injection valve,temperature regulated autosampler, column oven and Photodiode Arraydetector was used. The column was a Waters YMC 30, 5-micron, 4.6×250 mmwith a guard column of the same material. Standards were obtained fromDHI-Water & Environment, DK-2970 Horsholm, Denmark and Sigma ChemicalCo., St. Louis, Mo. 63178. The solvents for the mobile phase were 81methanol: 4 water: 15 tetrahydrofuran stabilized with 0.2% BHT.Injections were 20 μl. Separation was isocratic at 30° C. with a flowrate of 1.7 ml/minute. The peak responses were measured at 447 nm.

[0107] Using this protocol, the results from the first 507 samples werestatistically analyzed to establish average values for lutein andzeaxanthin content. To identify a mutant having a higher or lower thanaverage lutein and zeaxanthin concentration, a value of three standarddeviations greater than or less than the average was calculated. Thecalculated means, standard deviations and zeaxanthin ratios are shown inTable 4, below. TABLE 4 Lutein and Zeaxanthin Confidence IntervalCalculations Lutein Lutein + mg/g Zeaxanthin Zeaxanthin Fresh mg/g Freshmg/g Fresh Statistic Weight Weight Weight Ratio (%) Mean 0.64 0.04 0.685.98 Standard 0.14 0.01 0.147 1.1 deviation Mean + 1.06 0.07 1.12 9.28 3sd Mean − 0.22 0.007 0.24 2.68 3 sd

[0108] Based on the above values, samples were selected havingzeaxanthin ratios greater than 10 percent, combined lutein andzeaxanthin content greater than 1.12 mg/g fresh weight and combinedlutein and zeaxanthin content less than 0.24 mg/g fresh weight. A totalof 347 mutants were identified having a sum of lutein plus zeaxanthingreater than 1.12 mg/g, and 43 mutants having a zeaxanthin ratio greaterthan 10 percent were identified from 8192 samples using these selectionparameters. The total number of mutants resulting from each EMS seedtreatment is shown in Table 5, below. TABLE 5 Correlation of 13819Mutants to EMS Treatment 0.2% EMS 0.4% EMS Total Selection ParameterTreatment Treatment Plants Zeaxanthin 2 41 43 Ratio > 10% Lutein + 6 341347 Zeaxanthin > 1.12 mg/g dry weight Lutein + 2 175 177 Zeaxanthin <0.24 mg/g dry weight

[0109] Of the mutants having a zeaxanthin ratio greater than about 10percent zeaxanthin, about 47 percent had between 10 and under 13percent, whereas 53 percent exhibited 13 percent or greater.

EXAMPLE 5 Carotenoid Composition in Petals of Select Marigolds

[0110] Carotenoid compositions were determined for ‘Scarletade’wild-type and mutant samples selected from those identified in thescreening procedure described in Example 2. Petal samples were stored ina −80° C. freezer until mutants were identified. Samples werelyophilized, and the dried tissue was stored under argon at −80° C.until ready for analysis.

[0111] Extraction procedures were performed under red light. Driedpetals were ground to pass through a No. 40 sieve mesh size. A groundsample was accurately weighed and transferred into a 100 ml redvolumetric flask. To the sample, 500 microliters (μl) of H₂O were added,and the mixture was swirled for 1 minute. Thirty ml of extractantsolvent (10 ml hexane +7 ml acetone+6 ml absolute alcohol+7 ml toluene)were added, and the flask was shaken at 160 rpm for 10 minutes.

[0112] For saponification, 2 ml of 40% methanolic KOH were added intothe flask, which was then swirled for one minute. The flask was placedin a 56° C. H₂O bath for 20 minutes. An air condenser was attached toprevent loss of solvent. The sample was cooled in the dark for one hourwith the condenser attached. After cooling, 30 ml of hexane were added,and the flask was shaken at 160 rpm for 10 minutes.

[0113] The shaken sample was diluted to volume (100 ml) with 10% sodiumsulfate solution and shaken vigorously for one minute. The sampleremained in the dark for at least 30 minutes. A 35 ml aliquot wasremoved from the approximately 50 ml upper phase, and transferred to asample cup. An additional 30 ml of hexane were added into the flask thatwas then shaken at 160 rpm for 10 minutes. After approximately one hour,the upper phases were combined. For HPLC analysis, 10 ml aliquots weredried under nitrogen and stored under argon at −80° C.

[0114] HPLC equipment comprised an Alliance 2690 equipped with arefrigerated autosampler, column heater and a Waters Photodiode Array996 detector (Waters Corp., 34 Maple Street Milford, Mass. 01757).Separation was obtained with a YMC C₃₀ column, 3 μm, 2.0×150 mm with aguard column of the same material. Standards were obtained from ICCIndofine Chemicals Somerville, N.J. 088876 and from DHI-Water &Environment, DK-2970 Horsholm, Denmark.

[0115] The dried mutant samples were resuspended in tetrahydrofuran andmethanol to a total volume of 200 μl and filtered, whereas the controlwas not additionally concentrated. Carotenoids were separated using agradient method. Initial gradient conditions were 90% methanol: 5%water: 5% methyl tert-butyl ether at a flow rate of 0.4 milliliters perminute (ml/min). From zero to 15 minutes, the mobile phase was changedfrom the initial conditions to 80 methanol: 5 water: 15 methyltert-butyl ether, and from 15 to 60 minutes to 20 methanol: 5 water: 75methyl tert-butyl ether. For the following 10 minutes, the mobile phasewas returned to the initial conditions and the column equilibrated foran additional 10 minutes. The column temperature was maintained at 27°C. and the flow rate was 0.4 ml/minute. Injections were 10 μl. Themajority of peak responses were measured at 450 nm and additional areasadded from 286, 348, 400 and 472 nm extracted channels.

[0116] Values for carotenoid profiles of selected mutants are indicatedin Tables 6a, 6b and 6c, below, using peak area as percent of the totalarea. Indicated compound identifications are based on spectra extractedand maximal absorbance in ethanol (lambda maxima; ETOH) obtained formajor peaks in each chromatogram, some of which were verified byretention times of known standards. Values combine suspected isomers ofthe same compounds. Some compounds may contain minor impurities.Included in the Table are values for yellow colored American marigolds(yellow marigold) noted in Quackenbush et al., J. Assoc. Off. Anal.Chem., 55(3):617-621 (1972). Single entries are used in Tables 6a--6cfor neoxanthin/violaxanthin and chrysanthemaxanthin/flavoxanthincompound pairs that could not be separated by the procedure used here.TABLE 6a Relative Percent Distribution of Carotenoids in Petals ofTagetes erecta and Mutants Wave- length Marigold Selections in EtOHYellow Carotenoid (nm) Marigold ‘Scarletade’ 13819 117-185 124-257119-494 112-263 118-035 088-425 325-444 Phytoene 276, 286, 2.4 0.3 0.36.8 7.0 1.0 11.0 12.3 34.3 30.9 297 Phytofluene 331, 348, 2.6 0.5 0.44.0 4.2 0.9 7.5 7.4 17.8 13.3 (isomers) 367 ζ-Carotene 377, 399, nf* <0.1 <0.1 5.6 5.3 1.3 6.9 6.8 18.2 17.1 (cis/trans 425 isomers)Neurosporene 416, 440, nr** <0.1 <0.1 0.1 0.2 <0.1 <0.1 <0.1 3.5 3.5 470Lycopene 447, 472, nr   <0.1 <0.1 0.5 1.3 <0.1 <0.1 <0.1 1.0 2.8 504α-Carotene 423, 444, 0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.8 1.2 473β-Carotene 425, 451, 0.5 <0.1 <0.1 4.4 6.8 2.3 0.6 0.3 2.3 4.8 478Neoxanthin 415, 439, 0.8 467 Violaxanthin 419, 440, nr   1.5 4.1 13.312.8 16.7 4.3 3.5 0.7 1.1 470 Anthera- 422, 444, 0.1 3.1 5.5 12.5 14.419.2 4.1 4.5 0.9 1.5 xanthin 472 Lutein 420, 445, 72.3 84.9 81.7 13.31.3 <0.1 0.6 7.1 2.0 4.9 475 Zeaxanthin 428, 450, 16.4 4.7 5.9 21.3 30.635.7 16.5 18.2 2.0 4.0 478 α-Cryptoxanthin 421, 446, 0.8 <0.1 <0.1 <0.1<0.1 <0.1 32.2 26.9 <0.1 0.2 475 β-Cryptoxanthin 428, 450, 0.5 <0.1 <0.10.5 0.6 0.8 0.2 0.4 1.9 1.8 478 β-Zeacarotene 406, 428, 0.5 notidentified 454 Chrysanthema- 400, 421, 0.8 xanthin 448 Flavoxanthin 400,421, 1.3 <0.1 <0.1 2.3 1.5 4.5 0.8 0.5 0.2 0.2 448 Auroxanthin 380, 401,0.1 not identified 426 Other compounds that 0.8 5.0 2.1 15.3 14.0 17.615.1 12.0 14.3 12.7 show absorbance at 450 nm

[0117] TABLE 6b Relative Percent Distribution of Carotenoids in Petalsof Tagetes erecta and Mutants Wave- length Marigold Selections in EtOHYellow Carotenoid (nm) Marigold ‘Scarletade’ 13819 100-198 100-334100-470 101-190 114-315 Phytoene 276, 286, 2.4 0.3 0.3 4.8 3.9 6.1 3.45.2 (isomers) 297 Phytofluene 331, 348, 2.6 0.5 0.4 3.2 3.2 3.8 3.2 3.3(isomers) 367 ζ-carotene 377, 399, nf*  <0.1 <0.1 4.8 4.0 4.4 3.6 3.2(cis/trans 425 isomers) Neurosporene 416, 440, nr** <0.1 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 470 Lycopene 447, 472, nr   <0.1 <0.1 <0.1 <0.1 <0.1 <0.1<0.1 504 α-Carotene 423, 444, 0.1 <0.1 <0.1 0.3 0.4 0.2 0.4 0.2 473β-Carotene 425, 451, 0.5 <0.1 <0.1 0.8 0.7 0.5 0.8 0.5 478 Neoxanthin415, 439, 0.8 1.5 4.1 <0.2 0.3 <0.2 <0.2 <0.2 467 Violaxanthin 419, 440,nr   470 Anthera- 422, 444, 0.1 3.1 5.5 <0.2 <0.2 <0.2 <0.2 <0.2 xanthin472 Lutein 420, 445, 72.3 84.9 81.7 68.0 70.7 67.5 71.1 71.6 475Zeaxanthin 428, 450, 16.4 4.7 5.9 14.8 13.4 13.1 13.6 12.3 478α-Cryptoxanthin 421, 446, 0.8 <0.1 <0.1 0.6 0.6 0.5 0.6 0.4 475δ-Carotene 431, 456, nr   <0.1 <0.1 0.5 0.2 0.8 0.4 0.5 489 β-Crypto-428, 450, 0.5 <0.1 <0.1 <0.2 <0.2 <0.2 <0.2 <0.2 xanthin 478β-Zeacarotene 406, 428, 0.5 Not identified 454 Chrysanthema- 400, 421,0.8 <0.1 <0.1 <0.2 <0.2 <0.2 <0.2 <0.2 xanthin 448 Flavoxanthin 400,421, 1.3 448 Auroxanthin 380, 401, 0.1 Not identified 426 Othercompounds that 0.8 5.0 2.1 2.1 2.6 2.9 2.8 2.7 show absorbance at 450 nm

[0118] TABLE 6c Relative Percent Distribution of Carotenoids in Petalsof Tagetes erecta and Mutants Wave- length Marigold Selections in EtOHYellow Carotenoid (nm) Marigold ‘Scarletade’ 13819 126-415 098-240098-394 115-004 Phytoene 276, 286, 2.4 0.3 0.3 11.8 10.0 8.6 13.0(isomers) 297 Phytofluene 331, 348, 2.6 0.5 0.4 9.1 5.8 5.4 9.6(isomers) 367 ζ-Carotene 377, 399, nf*  <0.1 <0.1 5.0 3.6 3.5 10.3(cis/trans 425 isomers) Neurosporene 416, 440, nr** <0.1 <0.1 <0.1 <0.1<0.1 <0.1 470 Lycopene 447, 472, nr   <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 504α-Carotene 423, 444, nr   <0.1 <0.1 0.5 0.4 0.4 0.6 473 β-Carotene 425,451, 0.5 <0.1 <0.1 0.1 0.1 0.1 <0.1 478 Neoxanthin 415, 439, 0.8 1.5 4.10.3 0.4 0.4 <0.1 467 Violaxanthin 419, 440, nr   470 Anthera- 422, 444,0.1 3.1 5.5 1.7 1.9 2.2 1.9 xanthin 472 Lutein 420, 445, 72.3 84.9 81.761.7 70.1 71.0 52.3 475 Zeaxanthin 428, 450, 16.4 4.7 5.9 2.5 2.8 3.41.8 478 α-Crypto- 421, 446, 0.8 <0.1 <0.1 0.7 0.6 0.4 0.2 xanthin 475δ-Carotene 431, 456, nr   <0.1 <0.1 1.6 0.4 0.3 5.2 489 β-Cryptoxanthin428, 450, 0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 478 β-Zeacarotene 406, 428,0.5 Not identified 454 Chrysanthema- 400, 421, 0.8 <0.1 <0.1 <0.1 0.10.1 <0.1 xanthin 448 Flavoxanthin 400, 421, 1.4 448 Auroxanthin 380,401, 0.1 Not identified 426 Other compounds that 0.8 5.0 2.1 4.9 3.74.19 4.8 show absorbance at 450 nm

EXAMPLE 6 Carotenoid Composition in Leaves of Select Marigolds

[0119] Leaves of several marigold plants were assayed for the relativeconcentration of colored carotenoids present. Leaves from ‘Scarletade’and 13819 were used as controls for comparison to leaves from mutantplants. Assays were conducted as in Example 5 and are shown in Tables 7aand 7b, below, where single entries are used for neoxanthin/violaxanthinand chrysanthemaxanthin/flavoxanthin compound pairs that could not beseparted. Data in Tables 7a and 7b were collected from different groupsof plants grown under different conditions. TABLE 7a Relative PercentDistribution of Carotenoids in Leaves of Tagetes erecta and MutantsWave- length in Marigold Selections Carotenoid EtOH (nm) ‘Scarletade’13819 124-257 119-494 117-185 086-013 Phytoene 276, 286, 0.1 0.4 0.5 0.20.2 0.5 297 Neoxanthin 415, 439, 9.2 17.6 36.3 22.7 26.8 11.6 467Violaxanthin 419, 440, 470 Antheraxanthin 422, 444, 2.8 4.3 8.4 7.7 9.12.9 472 Lutein 420, 445, 44.3 37.8 0.5 <0.1 1.6 34.0 475 Zeaxanthin 428,450, 6.6 3.8 4.6 27.5 10.6 4.1 478 β-Carotene 425, 451, 22.6 26.5 34.125.0 32.7 35.8 478 α-Carotene 423, 444, 0.5 0.3 <0.1 <0.1 <0.1 0.2 473Chrysanthema- 400, 421, 1.1 1.0 0.9 4.1 3.2 0.5 xanthin 448 Flavoxanthin400, 421, 448 Other compounds that 12.8 8.3 14.7 12.7 15.8 10.4 showabsorbance at 450 nm

[0120] TABLE 7b Relative Percent Distribution of Carotenoids in Leavesof Tagetes erecta and Mutants Wave- length in Marigold SelectionsCarotenoid EtOH (nm) ‘Scarletade’ 100-198 100-334 100-470 101-190114-315 Phytoene 276, 286, Inadequate Peak Separation 297 Neoxanthin415, 439, 20.4 <0.1 0.3 <0.1 3.1 <0.1 467 Violaxanthin 419, 440, 470Antheraxanthin 422, 444, 1.6 1.7 1.8 1.6 5.4 1.1 472 Lutein 420, 445,48.3 24.7 27.6 28.8 27.7 24.3 475 Zeaxanthin 428, 450, 0.4 46.3 43.144.0 32.3 48.2 478 β-Carotene 425, 451, 15.9 14.5 17.3 14.5 19.6 13.8478 α-Carotene 423, 444, <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 473 Chrysanthema-400, 421, 1.0 <0.1 <0.1 <0.1 <0.1 <0.1 xanthin 448 Flavoxanthin 400,421, 448 β-Cryptoxanthin 428, 450, 0.3 0.3 0.3 0.6 0.3 0.9 478 Othercompounds that show 12.1 12.4 9.5 10.5 11.5 11.7 absorbance at 450 nm

[0121] Each of the patents and articles cited herein is incorporated byreference. The use of the article “a” or “an” is intended to include oneor more.

[0122] The foregoing description and the examples are intended asillustrative and are not to be taken as limiting. Still other variationswithin the spirit and scope of this invention are possible and willreadily present themselves to those skilled in the art.

What is claimed:
 1. Marigold flower petals, leaves or both flower petalsand leaves that contain one or more of an enhanced zeaxanthin ratio, anenhanced neoxanthin plus violaxanthin ratio, an enhanced β-caroteneratio, an enhanced α-cryptoxanthin ratio, an enhanced phytoene ratio oran enhanced phytofluene ratio compared to such a ratio present in theflower petals, leaves or both flower petals and leaves of a non-mutantmarigold wherein the xanthophylls are assayed as alcohols, the plantthat produced said petals or leaves having petals that contain ameasurable amount of zeta-carotene.
 2. The marigold flower petals,leaves or both flower petals and leaves according to claim 1 wherein thezeaxanthin ratio is greater than about 1:10.
 3. The marigold flowerpetals, leaves or both flower petals and leaves according to claim 2wherein said flower petals have a xanthophyll content of about 4 toabout 25 mg/g dry weight.
 4. The marigold flower petals, leaves or bothflower petals and leaves according to claim 1 wherein leaves exhibit anenhanced zeaxanthin ratio.
 5. The marigold flower petals, leaves or bothflower petals and leaves according to claim 4 wherein said leaves have axanthophyll content of about 0.2 to about 1.25 mg/g dry weight.
 6. Themarigold flower petals, leaves or both flower petals and leavesaccording to claim 1 wherein flower petals exhibit an enhancedzeaxanthin ratio.
 7. The marigold flower petals according to claim 6wherein zeaxanthin ratio is greater than about 2:10.
 8. Marigold flowerpetals that contain a ratio of zeaxanthin to zeaxanthin plus lutein ofgreater than about 2:10, said zeaxanthin and lutein being present in thepetals as fatty acid esters that are assayed as alcohols aftersaponification.
 9. The marigold flower petals according to claim 8 thatare from a hybrid.
 10. The marigold flower petals according to claim 8that are present in comminuted form.
 11. The marigold flower petalsaccording to claim 8 that further exhibit at least one of an enhancedneoxanthin plus violaxanthin ratio, an enhanced β-carotene ratio, anenhanced α-cryptoxanthin ratio, an enhanced phytoene ratio or anenhanced phytofluene ratio relative to a non-mutant marigold plant. 12.The marigold flower petals according to claim 8 wherein zeaxanthin ratiois greater than about 3:10.
 13. A marigold plant or regenerable portionthereof, whose flower petals, leaves or both flower petals and leavescontain one or more of an enhanced zeaxanthin ratio, an enhancedneoxanthin plus violaxanthin ratio, an enhanced β-carotene ratio, anenhanced α-cryptoxanthin ratio, an enhanced phytoene ratio or anenhanced phytofluene ratio compared to such a ratio present in theflower petals, leaves or both flower petals and leaves of a non-mutantmarigold wherein the xanthophylls are assayed as alcohols, the plantthat produced said petals or leaves having petals that contain ameasurable amount of zeta-carotene.
 14. The marigold plant orregenerable portion thereof according to claim 13 wherein the zeaxanthinratio of the flower petals is greater than about 1:10.
 15. The marigoldplant or regenerable portion thereof according to claim 13 wherein saidflower petals have a xanthophyll content of about 4 to about 25 mg/g dryweight.
 17. The marigold plant or regenerable portion thereof accordingto claim 13 wherein leaves exhibit an enhanced zeaxanthin ratio.
 18. Themarigold plant or regenerable portion thereof according to claim 13 thatfurther exhibits at least one of an enhanced neoxanthin plusviolaxanthin ratio, an enhanced β-carotene ratio, an enhancedα-cryptoxanthin ratio, an enhanced phytoene ratio or an enhancedphytofluene ratio compared to such a ratio present in the flower petals,leaves or both flower petals and leaves of a non-mutant marigold whereinthe xanthophylls are assayed as alcohols.
 19. The marigold plant orregenerable portion thereof according to claim 13 wherein the zeaxanthinratio is greater than about 2:10.
 20. The marigold plant or regenerableportion thereof according to claim 13 that is a hybrid.
 21. The marigoldplant or a regenerable portion thereof according to claim 13 wherein theregenerable portion is cells selected from the group consisting ofembryos, meristems, pollen, leaves, anthers, roots, root tips, andflowers, or protoplasts or callus derived therefrom.
 22. A marigoldplant or regenerable portion thereof, whose flower petals, leaves orboth flower petals and leaves contain a ratio of zeaxanthin tozeaxanthin plus lutein of greater than about 2:10, said zeaxanthin andlutein being present in the petals as fatty acid esters that are assayedas alcohols after saponification.
 23. The marigold plant or regenerableportion thereof according to claim 22 whose flower petals, leaves orboth flower petals and leaves further exhibits at least one of anenhanced neoxanthin plus violaxanthin ratio, an enhanced β-caroteneratio, an enhanced α-cryptoxanthin ratio, an enhanced phytoene ratio oran enhanced phytofluene ratio compared to such a ratio present in theflower petals, leaves or both flower petals and leaves of a non-mutantmarigold wherein the xanthophylls are assayed as alcohols.
 24. Themarigold plant or regenerable portion thereof according to claim 23whose flower petals, leaves or both flower petals and leaves exhibit aneoxanthin plus violaxanthin ratio of about 0.2 to about
 1. 25. Themarigold plant or regenerable portion thereof according to claim 23whose flower petals exhibit a β-carotene ratio of about 0.05 to about0.9.
 26. The marigold plant or regenerable portion thereof according toclaim 23 whose leaves exhibit a β-carotene ratio of about one.
 27. Themarigold plant or regenerable portion thereof according to claim 23whose flower petals exhibit a ratio of α-cryptoxanthin toα-cryptoxanthin plus lutein when assayed as alcohols aftersaponification that is about 0.25 to about 0.9.
 28. The marigold plantor regenerable portion thereof according to claim 23 whose flower petalsexhibit a phytoene ratio of about 0.3 to about
 1. 29. The marigold plantor regenerable portion thereof according to claim 23 whose flower petalsexhibit a phytofluene ratio of about 0.2 to about
 1. 30. A marigoldplant or regenerable portion thereof whose flower petals exhibit a ratioof α-cryptoxanthin to α-cryptoxanthin plus lutein when assayed asalcohols after saponification that is about 0.25 to about 0.9. 31.Pollen of the marigold plant of claim
 13. 32. An ovule of the marigoldplant of claim
 13. 33. A seed that on planting in a suitable environmentand growth to maturity yields a xanthophyll marigold plant according toclaim
 13. 34. A composition suitable for use as a food or feedsupplement that comprises a mixture of zeaxanthin and lutein dissolvedor dispersed in a comestible medium, wherein the zeaxanthin ratio isgreater than about 1:10.
 35. The composition according to claim 34wherein the zeaxanthin ratio is greater than about 2:10.
 36. Acomposition suitable for use as a food or feed supplement that comprisesa mixture of fatty acid esters of zeaxanthin and lutein dissolved ordispersed in a comestible medium, wherein the zeaxanthin ratio that isgreater than about 1:10.
 37. The composition according to claim 36wherein the zeaxanthin ratio is greater than about 2:10.
 38. Thecomposition according to claim 36 wherein said comestible medium is anedible oil.
 39. The composition according to claim 36 wherein saidcomestible medium is a pharmaceutically acceptable binding agent.
 40. Anoleoresin of a mutant marigold, said oleoresin having one or more of anenhanced zeaxanthin ratio, an enhanced neoxanthin plus violaxanthinratio, an enhanced β-carotene ratio, an enhanced α-cryptoxanthin ratio,an enhanced phytoene ratio or an enhanced phytofluene ratio relative toan oleoresin from a non-mutant marigold.
 41. The oleoresin according toclaim 40 that contains free xanthophylls, carotenes and chlorophyll. 42.The oleoresin according to claim 40 that contains xanthophyll esters,carotenes and is largely free of chlorophyll.
 43. Marigold flower petalsthat contain colored carotenoids comprised of α-cryptoxanthin.
 44. Themarigold flower petals according to claim 43 wherein α-cryptoxanthin isthe most abundant carotenoid present in the flower petals.